Trichoderma reesei is a filamentous fungus able to carry out efficient hydrolysis of crystalline cellulose to glucose through the action of a number of secreted cellulase enzymes. The major secreted protein is cellobiohydrolase I (CBHI) that may constitute up to 60% of the secreted proteins. This amount, derived from the single copy cbh1 gene, corresponds to roughly 25% of all protein synthesized by the fungus in cellulase-inducing growth conditions.
Availability of the carbon source regulates production of CBHI (reviewed in Bisaria, V. S. et al., CRC Crit. Rev. Biotechnol. 9: 61-103 (1989)) and the regulation occurs at the level of transcription of the gene encoding CBHI (E1-Gogary, S. et al., Proc. Natl. Acad. Sci. USA 86: 6138-6141 (1989); Fowler, T. et al., Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases and other hydrolases, Espoo, Finland. Suominen P., and Reinikainen T. (eds.), Foundation for Biotechnical and Industrial Fermentation Research 8: 199-210, Helsinki (1993); Kubicek, C. P. et al., Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases and other hydrolases, Espoo, Finland. Suominen P, and Reinikainen T (eds.), Foundation for Biotechnical and Industrial Fermentation Research 8: 181-188, Helsinki (1993); Penttila, M. E. et al., Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases and other hydrolases, Espoo, Finland. Suominen, P., and Reinikainen, T. (eds), Foundation for Biotechnical and Industrial Fermentation Research 8: 189-198, Helsinki (1993): Abrahao-Neto, J. et al., Biochemistry 34: 10456-10462 (1995)).
cbh1 mRNA has been shown to be several thousand fold more abundant when the fungus is grown on a medium containing cellulose compared with glucose. Use of glucose as a carbon source represses cellulase expression (Penttila, M. E. et al., Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases and other hydrolases, Espoo, Finland. Suominen P, and Reinikainen T (eds. ), Foundation for Biotechnical and Industrial Fermentation Research 8: 189-198, Helsinki (1993)).
Sophorose consists of two glucose units linked by a .beta.-1,2-glycosidic bond. Sophorose has also been considered to be a possible natural inducer formed from .beta.-1,4-linked cello-oligosaccharides such as cellobiose by transglycosylation (Vaheri, M. et al., Biotechnol Lett. 1: 41-46 (1979); Gritzali, M. etal., Adv. Chem. Ser. 181: 237-260 (1979); Loewenberg, J. R., Arch. Microbiol. 137: 53-57 (1984)). Sophorose is known to induce the cbh1 promoter.
Successful production of recombinant proteins of fungal and mammalian origin under the control of the cbh1 promoter has been achieved (reviewed by Paloheimo, M. et al., Proceedings of the second TRICEL symposium on Trichoderma reesei cellulases and other hydrolases, Espoo, Finland. Suominen P, and Reinikainen T (eds.), Foundation for Biotechnical and Industrial Fermentation Research 8: 229-238, Helsinki (1993); Keranen, S. et al., Curr. Opin. Biotechnol. 6: 534-537 (1995)). Despite biological and biotechnical importance of cellulase production, the specific elements regulating such expression at the transcriptional level are generally unknown. Promoter sequence comparisons carried out for fungal cellulase promoters (Raguz, S. et al., Gene 119: 183-190 (1992); Stangl, H. et al., Curr. Genet. 23: 115-122 (1993); Yague, E. et al., Mol. Microbiol. 12: 41-47 (1994)) have revealed little similarities and the functional significance of this similarity, if any, is unknown. The analysis of the promoter of the cellulase gene cbh2 of T. reesei by a preliminary gel shift analysis showed that cellular proteins bind the promoter sequences (Stangl, H. et al., Curr. Genet. 23: 115-122 (1993)). However, the function of those proteins, whether they were involved in the regulatory induction of transcription by specific activators or were generally required for the transcriptional process (such as a polymerase), is also unknown.
The cbh1 promoter is contained on a long 2.2 kb piece of T. reesei genomic DNA flanked on the 5' end by an EcoRI site and on the 3' end by the start of the CBHI coding sequence. The ability to design recombinant constructs and vectors for the production of a desired sequence under the control of this promoter would be more efficient if it were possible to shorten the promoter but yet retain the ability of the promoter to respond to inducers such as sophorose. This is especially desirable when the vector is a shuttle vector that is to be maintained in a bacterial or yeast host. However, such shortened forms of the cbh1 promoter are unknown.